Quantification of quaternary structure stability in aggregation-prone proteins under physiological conditions: the transthyretin case.
Quantification of quaternary structure stability in aggregation-prone proteins under physiological conditions: the transthyretin case.
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DOI：
10.1021/bi500739q
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发表时间：
2014-10-21
期刊：
影响因子：
2.9
通讯作者：
Reixach, Natalia
中科院分区：
文献类型：
--
作者：
Robinson, Lei Z.;Reixach, Natalia
蛋白质的四级结构稳定性通常在方便的实验室时间尺度上加速其聚集/展开过程的条件下进行研究。这些条件包括高温或高压、离液剂介导的解折叠、或低或高pH。这些方法具有非生理性的局限性，并且溶液中蛋白质的浓度随着反应的进行而变化。我们描述了一种方法来定义四级结构的稳定性淀粉样蛋白同源四聚体蛋白转甲状腺素蛋白（TTR）在生理条件下。该方法从基于TTR与串联标记的（FT 2）TTR对应物的亚基交换速率的测量的描述的方法扩展。我们证明TTR与FT 2·TTR的亚基交换可以使用半天然聚丙烯酰胺凝胶电泳技术进行分析和定量。此外，我们还对两种来源于野生型的FT 2·TTR变体和淀粉样蛋白生成变体Val 122 Ile TTR进行了生物病理学特征分析，这两种变体均与生命后期的心脏淀粉样蛋白沉积相关。FT 2·TTR变体与其未标记的对应物相比具有相似的淀粉样蛋白生成潜力和相似的热力学和动力学稳定性。我们利用该方法研究了小分子SOM 0226（一种正在临床开发的用于预防和治疗TTR淀粉样变性的再利用药物）稳定TTR的潜力。结果使我们能够表征SOM 0226与TTR的结合能。所描述的技术非常适合于在生理条件下研究其他人类聚集倾向蛋白质的四级结构。
The quaternary structure stability of proteins is typically studied under conditions that accelerate their aggregation/unfolding processes on convenient laboratory time scales. Such conditions include high temperature or pressure, chaotrope-mediated unfolding, or low or high pH. These approaches have the limitation of being nonphysiological and that the concentration of the protein in solution is changing as the reactions proceed. We describe a methodology to define the quaternary structure stability of the amyloidogenic homotetrameric protein transthyretin (TTR) under physiological conditions. This methodology expands from a described approach based on the measurement of the rate of subunit exchange of TTR with a tandem flag-tagged (FT2) TTR counterpart. We demonstrate that subunit exchange of TTR with FT2·TTR can be analyzed and quantified using a semi-native polyacrylamide gel electrophoresis technique. In addition, we biophysically characterized two FT2·TTR variants derived from wild-type and the amyloidogenic variant Val122Ile TTR, both of which are associated with cardiac amyloid deposition late in life. The FT2·TTR variants have similar amyloidogenic potential and similar thermodynamic and kinetic stabilities compared to those of their nontagged counterparts. We utilized the methodology to study the potential of the small molecule SOM0226, a repurposed drug under clinical development for the prevention and treatment of the TTR amyloidoses, to stabilize TTR. The results enabled us to characterize the binding energetics of SOM0226 to TTR. The described technique is well-suited to study the quaternary structure of other human aggregation-prone proteins under physiological conditions.
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影响因子：
17.1
作者：
Alhamadsheh MM;Connelly S;Cho A;Reixach N;Powers ET;Pan DW;Wilson IA;Kelly JW;Graef IA
通讯作者：
Graef IA
DOI：
10.1073/pnas.1121005109
发表时间：
2012-06-12
影响因子：
11.1
作者：
Bulawa, Christine E.;Connelly, Stephen;Labaudiniere, Richard
通讯作者：
Labaudiniere, Richard
影响因子：
2.9
作者：
COLON, W;KELLY, JW
通讯作者：
KELLY, JW
影响因子：
2.9
作者：
Hurshman, AR;White, JT;Kelly, JW
通讯作者：
Kelly, JW
影响因子：
2.9
作者：
Hammarström, P;Jiang, X;Kelly, JW
通讯作者：
Kelly, JW